Saccharides, which are also called sugars or carbohydrates, are major components of biological systems. Saccharides make up about 80% of the dry weight of plants, and are essential constituents of metabolic pathways in higher animals, either as monomers (monosaccharides) or as polymers which are composed of covalently linked monosaccharides (oligosaccharides). In addition, saccharides are often found as components of larger biological macromolecules, including proteins, lipids and nucleic acids. In this wide variety of forms, saccharides have a large number of critical functions in nature.
Because of the importance of saccharides in biological systems, procedures for unambiguous identification of monosaccharides, both free and as monomeric constituents of oligosaccharides, are of considerable utility. Furthermore, procedures for selectively identifying the monosaccharide at the end of an oligosaccharide are important for proof of polymeric saccharide structures.
Most monosaccharides in nature have a backbone structure that contains five carbon atoms (pentoses) or six carbon atoms (hexoses). In the linear form of these molecules, each backbone carbon atom may be covalently bonded to an oxygen atom, to form a series of hydroxyl groups (or modifications thereof) and a ketone or aldehyde group. Those molecules that contain an aldehyde group are referred to as aldoses, whereas those that contain a ketone group are known as ketoses.
In nature, most monosaccharides are aldoses. While members of each class of aldoses (e.g., pentoses and hexoses) are structurally similar, numerous distinct stereoisomers exist within each class. For example, in the hexose series, there are sixteen hexoses having an aldehyde group at C-1 and hydroxyl groups at all other positions along the carbon backbone. These sixteen stereoisomers are shown in FIG. 1 in their linear (Fischer projection) forms. (The Fischer projection represents the molecules so that the hydrogen atom and hydroxyl group on an asymmetric carbon atom come out of the plane of the paper towards the reader, as shown in the first molecule of FIG. 1.) Each stereoisomer is a different molecule, varying only in the stereochemistry at the different asymmetric carbon atoms.
Ketoses are also found naturally, usually having a ketone at the C-2 position (i.e., the second carbon of the carbon backbone) and hydroxyl groups at all other carbons, as shown below for D-fructose (the backbone carbons are designated as 1-6). ##STR1## Ketoses may also have different numbers of carbons, there being, for example, hexoses and pentoses having a ketone function at C-2. In the hexose series of ketoses, there are eight stereoisomers having a ketone group at C-2. These hexoses are the D and L forms, respectively, of fructose, sorbose, tagatose, and psicose, each of which varies in the stereochemistry at the asymmetric carbons C-3 to C-5.
In addition to these variations in stereochemistry, hexoses and pentoses are also capable of existing in five-atom ring forms (also known as five-membered rings or furanoses) and six-atom ring forms (also known as six-membered rings or pyranoses). In these ring forms, an oxygen atom is present in the ring structure. For example, shown below are the five-membered and six-membered rings for D-glucose. ##STR2## In solution, aldose and ketose monosaccharides exist in equilibrium between the ring forms and the open chain forms, as shown in FIG. 1. Depending on the arrangement of the substituents at C-1, the ring form may be the .alpha. or the .beta. anomer, as shown in the equilibrium for glucose below. ##STR3## Monosaccharides that are capable of interconverting in solution to give the open chain forms may themselves be reduced (i.e., the carbonyl group converted to a lower oxidation state such as an alcohol), and are therefore referred to as reducing monosaccharides. Both free monosaccharides and monosaccharides located at one end (the reducing end) of an oligosaccharide may be reducing monosaccharides.
This large number of distinct monosaccharides that have the same elemental composition creates problems for identification of particular saccharides. For example, each of the 16 hexoses that have an aldehyde group at C-1 can exist in five forms (four cyclic forms and the linear form), which rapidly interconvert in solution. Consequently, during a chromatographic analysis, there are 16.times.5=80 possible forms of very closely related, interconverting molecules, rendering the chromatograms complex and often impossible to analyze.
Further complicating the monosaccharide analysis is the large number of such molecules that exist in nature which are modified in some way from the basic "core" structures discussed above. For example, there are common and abundant monosaccharides from many biological sources that lack one or more hydroxyl groups along the carbon backbone (termed deoxy-sugars if one hydroxyl group is lacking or dideoxy-sugars when two such groups are lacking), that contain an ether linkage instead of a hydroxyl group (usually a methyl ether, and termed methylated sugars), that have an amino group or N-acylamino group replacing a hydroxyl group (termed, respectively, amino sugars or acylamino sugars), or that have a carboxylic acid group instead of a CH.sub.2 OH group at C-6 (termed uronic acids). Monosaccharides may also be branched, rather than having a linear carbon backbone (termed branched chain sugars). Hamamelose, shown in FIG. 2, is an example of a branched monosaccharide. In rare cases, monosaccharides may include combinations of these modifications, or may contain other modifications including, but not limited to, esters, cyclic acetals, sulfate esters and phosphate esters. Examples of some typical modifications are provided in FIG. 2.
Many monosaccharides are linked together to form oligosaccharides. In linear oligosaccharides, the monosaccharides are always linked together with the linkage from C-1 of one monosaccharide to one of C-2, C-3, C-4, C-5 or C-6 of another monosaccharide. For example, shown below is an oligosaccharide in which one D-glucose ("Glc") molecule is linked to another D-glucose molecule from C-1 of the D-glucose on the left side to C-4 of the D-glucose on the right side. ##STR4##
The bond between the two D-glucoses is termed a "glycosidic bond." The linkage between the two glucose molecules may be .alpha. or .beta., depending on the arrangement of the substituents at C-1 of the D-glucose on the left. The D-glucose on the right side of the di-glucose oligosaccharide structure possesses a hydroxyl group (at C-1) which may exist as an aldehyde group in the open chain form of that monomer. Therefore, the D-glucose on the right is the reducing monosaccharide. Conversely, the D-glucose on the left side of the structure does not possess a hydroxyl group at C-1 and, therefore, is termed a non-reducing monosaccharide.
Oligosaccharides contain no more than one reducing monosaccharide. In oligosaccharides that are linear (i.e., monosaccharides that are linked in a straight chain without branching), there will be one reducing end and one non-reducing end. If there is branching in an oligosaccharide (i.e., more than one monosaccharide is linked to a given monosaccharide), there will still be only one reducing end but two or more non-reducing ends. Since each monosaccharide may be linked to different positions of the adjacent monosaccharide, there is the potential for oligosaccharides of significant complexity. However, each reducing oligosaccharide possesses only one reducing monosaccharide (which may be an aldose or a ketose) which can exist in the open-chain form. As a result, there is the potential for unambiguous identification of the reducing aldose or ketose monosaccharide using reactions that modify only the carbonyl group on that monosaccharide.
Unambiguous identification of monosaccharides (either free or at the reducing end of oligosaccharides) has, however, proven to be difficult. Because of the enormous number of existing, distinct monosaccharides (many of which have similar or identical migration properties in chromatography) and the different interconverting forms of each monosaccharide, identification of a monosaccharide cannot be achieved with certainty based solely on its retention time in a chromatographic system. The use of mass spectrometry to identify monosaccharides is therefore essential, because the mass fragmentation spectral patterns (for example in electron impact mass spectrometry) clearly distinguish among the above classes of molecules. As long as all possible members of a class that give the same mass spectrum (such as, for example, all of the hexoses) are separable, the use of mass spectrometry permits monomers to be identified without doubt.
To limit the number of possible forms in which a monosaccharide may exist, and thus to improve the separation of members of each class by chromatography prior to mass spectrometry, derivatization techniques are often employed. Some derivatizations, however, can also result in cyclic products. Since there are usually four such products (.alpha. and .beta. furanose and .alpha. and .beta. pyranose derivatives), each monosaccharide can give rise to four chromatographic peaks in varying quantities. This makes analysis of complex mixtures difficult, if not impossible, due to the abundance of overlapping peaks.
Some modifications of monosaccharides that generate a single chromatographic peak have been described. The most useful of these convert the monosaccharide to a linear derivative that is incapable of cyclizing, and which therefore does not interconvert during chromatography. One procedure which has been employed extensively in the past involves the direct reduction of a monosaccharide aldehyde or ketone group to a hydroxyl group. This reaction converts an aldose or ketose to an alditol. Peracylation of alditols results in derivatives that are suitable for gas chromatography-mass spectrometry (GCMS). However, this procedure suffers from a serious limitation because some alditols may be generated from more than one monosaccharide. For example, as shown in FIG. 3, the same alditol product is generated by reduction of the molecules D-glucose and L-gulose. As a result, this procedure cannot distinguish between those two stereoisomers. Additional pairs of aldoses which give the same alditol product are L-glucose and D-gulose, D-altrose and D-talose, L-altrose and L-talose, D-galactose and L-galactose, and D-allose and L-allose. Among the aldoses, only D-mannose, L-mannose, D-idose and L-idose generate unique alditol products.
In addition, the reduction of a ketose generates a pair of alditols. For example, D-fructose generates D-glucitol and D-mannitol, D-sorbose generates D-gulitol and D-iditol, D-tagatose generates D-galactitol and D-talitol and D-psicose generates D-allitol and D-altritol. Thus, the formation of a given alditol upon reduction of a monosaccharide does not unambiguously identify the monosaccharide, since both ketoses and aldoses may generate the same alditols.
Other methods have been described for monosaccharide analysis that result in acyclic derivatives. For GCMS, the most useful derivatives for quantitation of monosaccharides are the aldononitriles (which may be generated from aldoses) and dithioacetals (which may be generated from aldoses and ketoses), since methods exist that achieve essentially quantitative yields of these derivatives. Representative examples of these derivatives are given in FIG. 4. These derivatization techniques have the important advantage that they yield a single product for each monosaccharide. For example, each of the 16 different aldose hexoses gives a unique derivative. Prior to analysis by GCMS, the aldononitrile derivatives are normally converted to acetyl esters and the dithioacetals are normally converted to trimethyl silyl ethers or trifluoroacetyl esters. The acetyl esters are stable, but the trimethyl silyl esters and trifluoroacetyl esters must be analyzed immediately after preparation and are very susceptible to cleavage by atmospheric water prior to and during analysis.
In addition, these derivatives cannot be used for analysis of a monosaccharide at the reducing end of an oligosaccharide. The synthesis of dithioacetals will, itself, cleave the glycosidic linkages, resulting in nonselective derivatization of the monosaccharide components of the oligosaccharide. Furthermore, dithioacetals are not stable under the acidic conditions required to cleave glycosidic linkages in oligosaccharides. While the formation of the aldononitrile may be achieved without cleavage of the glycosidic linkages, this derivative does not survive the acidic conditions needed to cleave the derivatized monosaccharide from the oligosaccharide.
Accordingly, there is a need in the art for a method of generating derivatives of free monosaccharides, as well as monosaccharides at the reducing end of an oligosaccharide, where the derivatives (1) permit unambiguous identification of each former monosaccharide, (2)are stable at room temperature and below for extended periods (i.e., more than one year), (3) survive conditions that cleave glycosidic linkages, thereby permitting identification of the monosaccharide at the reducing end of an oligosaccharide and (4) are amenable to on-line mass spectral analysis in techniques such as, but not limited to, GCMS, LCMS (liquid chromatography-mass spectrometry) and CZE (capillary zone electrophoresis-mass spectrometry), or are amenable to multiple fragmentation of daughter ions in procedures such as, but not limited to, GC-MSMS or GC-MSMSMS.
The present invention fulfills these needs and provides further related advantages.